Development of a Chromatographic Method to Authenticate Aspirin Brands

Counterfeit pharmaceuticals pose a threat to society that can include inaccurate amounts of the active pharmaceutical ingredient (API), no API, or containing off-target compounds. For example, there are many recent examples of counterfeit pharmaceuticals containing potentially lethal doses (\u3e 2 mg) of fentanyl (i.e., a synthetic opioid). Current measures to combat illicit pharmaceuticals (e.g., unique packaging and product serialization) have merit, however with evolved technologies, counterfeiters can relatively easily simulate these measures and continue to distribute illicit pharmaceuticals. The only accurate way to definitively determine that a suspected counterfeit is, in fact, counterfeit is advanced chemical analysis. However, current methods of authentication via chemical analysis have disadvantages. Therefore, a general drug authentication method was developed to authenticate and correctly classify pharmaceuticals, specifically Bayer®, Walgreens©, and Premier Value® aspirin. Gaschromatography mass-spectrometry (GC-MS) and liquid-chromatography tandem mass spectrometry (LC-MS/MS) were evaluated for analysis of aspirin. LC-MS/MS produced the most consistent analysis results. Additionally, three statistical techniques, linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), and atypicality analysis, were evaluated for their usefulness in source attribution. LDA outperformed the other statistical treatments, with perfect classification of the training data set using LDA. However, when applying the method to a set of double-blinded pills, all statistical treatments failed to correctly classify over 25% of the pills. Because this method of source attribution was inconsistent, further optimization of the method is needed before introducing unknown sources

Anthropogenic Organic Compound Interactions with Humic Materials

Humic acid, fulvic acid, and humin were isolated from soil by utilizing the traditional alkali method. The association of selected AOC (anthropogenic organic compounds) with humic materials was studied. A major emphasis of this research was elucidate and understand how humic and fulvic acid interact with AOCs. The major thrust of this research was the hypothesis that humic acid does, in fact, form an organized aggregate known as a micelle. This hypothesis was then applied to the lowest molecular weight fraction of humic material-fuvic acid. Another emphasis of this work was to probe the interactions of AOCs with humin. The actual humin fraction was extracted to determine the role of the lipid and brominate-treated to examine the role of organic matter for AOC interactions with humin. Three experimental techniques were employed to determine whether humic and fulvic acid micelle formation is an accurate description of the aggregation process that may be occurring; they are surface tension, solubility measurements utilizing radio-labeled AOCs, and small-angle X-ray scattering spectrophotometry. The results obtained indicated there was not substantial aggregation due to classical micelle formation when the concentration of humic acid was increased. However, this evidence suggested that as the concentration of humic acid increased to a critical point, the humic acid components arrange themselves by directing the hydrophobic portions of the humic acid toward the center of the aggregate. The hydrophobic center of the humic acid, at higher concentrations, was able to substantially solubilize otherwise hydrophobic (low water solubility) AOCs compared to the AOC water solubility. This observation has been reported recently in the literature as the formation of a pseudomicelle of hemimicelle. The ability of humic and fulvic acid to increase the water solubility of DDT, naphthalene, and pyrene with respect of pH and ionic strength was also investigated, and the implications are discussed. The interaction of several AOCs with humin was studied to determine if the partitioning model was considered valid for humin. The humin treated to remove the lipid and organic matter to determine the effects of AOC adsorption to the altered humin. The determination of the amount of AOC that associated (sorbed) to the unaltered humin, extracted lipid humin, and organic free humin was determined utilizing a low pressure liquid chromatograph column. One criterion supporting the partitioning model is the lack of competitive effects upon adsorption with more than one AOC. To validate the partitioning model, the surface area of the sorbent should be lower after adsorption. This was accomplished using Brunauer, Emmett and Teller (BET) surface analyses. The surface areas of the samples were determined before and after application of the AOC. The surface area results and sorption results of humin with AOCs are presented and the implications to soil are addressed

A Kinetic Study of the Reactivity of Various Cytochrome C Systems with Cobalt Phenanthroline and Cobalt Oxalate

Cytochrome c ( cyt c) is a heme containing protein of the mitochondrial electron transport chain. Cyt c transfers electrons from cytochrome reductase to cytochrome oxidase. The reactivity of cyt c with the inorganic reagents, cobalt phenanthroline, (Co(phen)33 +), and cobalt oxalate, (Co(oxh3-) was studied. The effects of ionic strength, pH, amino acid side chain modifications to cyt c, and association of cyt c with synthetic vesicles on the reactivity of cyt c have been determined. Generally, oxidation of cyt c associated with membranes by Co(phen)3(Cl04)3 is faster than solution cyt c. The trend is reversed for the oxidation of cyt c by K3Co(ox)3; the rate is slower when associated with membranes. The activation enthalpy is larger for the oxidation of cyt c by Co(phen)3 3 + than Co(ox)l-. The activation entropy is more negative for the oxidation of cyt c by Co(ox)?- than Co(phen)3 3+ . Photooxidation, chloramine T oxidation, and sulfonyl chloride modification of cyt c reveal that Met-80, Met-65 and Lys-79 are important in regulation of the open and closed forms of the protein. Photomodification of cyt c yields a protein whose reactivity is similar to native cyt c, however the association of PM cyt c with vesicles yields a system that is much more reactive than vesicle-bound native cyt c. The chloramine T modification of cyt c to a sulfoxide at both Met-65 and 80 destroys the ability of the protein to open and close substantiating that Met-65 is located in the hinge region and is important for conformational changes in the protein. The EPR spectra of chloramine T modified cyt c show the loss of Met-80 giving an open heme crevice and a new high-spin form. The sulfonyl chloride modifications yielded proteins whose reactivity toward Co(ox)l- and Co(phen)3 3+ are essentially the same as native cyt c. The EPR spectra of the sulfonyl chloride derivatives display a strong free radical signal due to the oxidation of the modifying fluorophore

Atypicality Based Measures for the Identification of Counterfeit Aspirin

In this work we are focused on building a pattern recognition system for chemometric data arising from the LC-MS.MS spectrometry analysis of brand name aspirin. Originally, the goal of the work was to build a set of discriminate functions that can separate between known brands of manufactured aspirin, similar to LDA, which finds a set of projections that optimize linear separation relative to within class variation [1]. However, these functions must assign each observation to a known class, even if the likelihood of an observation having arisen from any given class is very small. As an alternative, we investigate the use of atypicality measures as a way around this issue. The atypicality of an observation with respect to a given population (or class) is the chance of drawing a new sample from a given class that has a greater likelihood of being observed than the actual observation that we are considering assigning to said class. A pattern recognition system based off atypicality measures would assign an observation to the class with the smallest atypicality given that it is not above some threshold. This threshold can be considered a method for determining if an observation is likely to not belong to any known classes at all. We will perform a simulation study comparing the effectiveness of atypicality based methods to LDA and QDA methods when the assumptions of the discriminate functions are satisfied, and then apply the three methods to a chemometric data set related to the analysis of aspirin pills